Division of labor in trypanosome RNA processing and export through expanded Mex67 paralogs

Figure 1.. Affinity capture of the TbMtr2 and identification of new Mex67 paralogs. (A) Using the GFP tagged TbMtr2 (marked with a ‡), under high stringency biochemical conditions (Buffer 1), we affinity isolated structural components of the NPC (sky blue), transport factors connected to the GTPase Ran cycle (orange), and TbMex67 (navy blue) with which TbMtr2 is known to form a heterodimer (38). In addition, we discovered interaction with two putative TbMex67 paralogs which we termed TbMex67b and TbMex67L (for like or large) denoted in navy blue like TbMex67. Under low stringency conditions (Buffer 2), we affinity isolated the entire trypanosome NPC as well as exportin 7 (Xpo7), the RNA export component Gle2 (85–87) and the lamin proteins NUP-1 and NUP-2 (54 133), and a hypothetical protein (gray). Affinity isolates were resolved by SDS–PAGE and visualized by Coomassie staining. Proteins bands were excised and identified by mass spectrometry. (B) A schematic of the domains that are present on each protein and highlights the difference between them. (C) An illustration of the differences between the architecture and domains of the three TbMex67 paralogs as predicted by AlphaFold (76). The…

Figure 2.. GFP tagging of the TbMex67 paralogs and their protein interactomes. (A) We in situ GFP tagged TbMex67, TbMex67b, and TbMex67L at their chromosomal loci in both the PCFs that infect tsetse flies and the BSFs that infect vertebrates. The TbMtr2-GFP from PCFs is shown for comparison. We found that except for TbMex67L, other paralogs and TbMtr2 localize to NPCs at the nuclear envelope as well as the nucleoplasm albeit predominantly around the nucleolus. (B) We performed affinity capture using each GFP tagged paralog in PCFs and BSFs (marked with a ‡) and discovered that TbMex67 and TbMex67b interact predominantly with the NPC, whilst TbMex67L interacts only with ribosome biogenesis proteins, possibly a reflection of its nucleolar only localization. However, there is a significant difference between the interactomes of TbMex67 and TbMex67b in BSFs, with TbMex67b forming additional interactions with TbSub2, HYP, and PABP2, suggesting it may operate in a pathway that distinguishes it from TbMex67 in BSFs.

Figure 3.. RNA interference knockdown of the Mex67 paralogs and poly(A) FISH to determine role in RNA export. To assess the role of the TbMex67 paralogs on mRNA export, we performed RNA FISH after induction of RNAi silencing for each individual paralog. There was a rapid loss of fitness in BSFs, accompanied by accumulation of mRNA in the nucleus of TbMex67, and in and around the nuclei of TbMex67b cell lines, but not TbMex67L indicating it is not involved in mRNA export. In PCFs, there was a rapid loss of fitness in TbMex67 cell lines post RNAi induction and accumulation of mRNA in the nucleus, but not in the TbMex67b cell lines which are less affected by RNAi ablation of the TbMex67b RNA. The RNAi KD is extremely poor (5%) in these cell lines. RNAi loss of fitness was very gradual post RNAi induction in TbMex67L cell lines. However, unlike in BSFs, over time, there is a gradual halo-like mRNA accumulation around the nuclei of the TbMex67L cell line. We are investigating TbMex67b and TbMex67L further.

Figure 4.. Affinity capture and identification of the RNA cargo of each TbMex67 paralogs. We affinity captured each TbMex67 paralog in PCFs and BSFs, then extracted interacting RNA and performed Illumina sequencing to determine which RNA species each paralog interacts with. Results were plotted on a linear scale (main graph), as well as in log10 (inset graph). In all the graphs, the X-axis represents TbMex67, which has been characterized in various studies, whilst the Y-axis represents either TbMex67b or TbMex67L, both of which had not yet been characterized prior to this study. The gray points on each graph represent transcripts that are not differentially enriched with statistical significance (P > 0.01). (A) TbMex67 (blue) and TbMex67b (red) share common mRNA cargo in both BSFs and PCFs, with a subset of mRNA species enriched in TbMex67 or TbMex67b, with highly abundant transcripts such as elongation factor 1-alpha (EEF1A) and heat shock protein (HSP70) in PCFs and calpain cysteine peptidase (ClpCysPep) in BSFs for TbMex67b. (B) There is a significant enrichment of mRNA encoding RBPs and zinc finger proteins on the BF TbMex67 mRNA cargo as compared with TbMex67b, suggesting a possible…

Figure 5.. TbMex67L is involved in 60S (LSU) maturation. (A) Polysomes were prepared at 18hrs to avoid pleiotropic effects associated with dying cells, from Control (−Tet) and RNAi cells (+Tet) for TbMex67 and TbMex67L RNAi cell lines. RNAi KD of TbMex67L results in a drop in the 60S peak and the appearance of shoulders (halfmers) on the polysome profile of cells depleted of TbMex67L, but not TbMex67 showing that this phenotype is specific for TbMex67L. (B) A schematic of the trypanosome rRNA processing pathway. The 28S rRNA is fragmented into two large and four small fragments unlike in opisthokonts. (C) In both BSFs and PCFs affinity isolated RNA, the LSU is still connected to the 5.8S, M1, M2 as well as ITS2, 3, 4, and 5, suggesting TbMex67L associates with LSU rRNA that is still being processed. Additionally, there is an association of TbMex67L with SSU rRNA in BSFs parasites and not insect stage PCF parasites that is apparent in Fig. 4.